In vitro synthesis of the membrane-bound D-lactate dehydrogenase of Escherichia coli

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Abstract
Synthesis of the membrane-bound flavin-linked D-lactate dehydrogenase of E. coli was studied by using a recombinant plasmid containing the dld gene. Expression of the cloned dld gene was achieved in vivo with transformed minicells or in vitro with a fractionated transcription/translation system. In both instances, a product is observed that is specifically immunoprecipitated by .gamma.-globulin prepared against the purified enzyme and comigrates with authentic D-lactate dehydrogenase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The product is catalytically active and binds to membrane vesicles during or after synthesis. It seems likely that the protein is synthesized in mature form and binds to the membrane without a leader peptide sequence. Addition of FAD to the in vitro reaction mixtures causes a 2-fold increase in the synthesis of the enzyme, suggesting that the cofactor plays a regulatory role in the synthesis of the apoprotein. L factor, a protein involved in regulation of protein elongation, has an inhibitory effect on the expression of the dld gene and a stimulatory effect on the expression of the ndh gene (encoding NADH dehydrogenase).